Polyfunctional radical scavenger hydrogel formulation

ABSTRACT

A polyfunctional radical scavenger hydrogel formulation providing extended protection of the extracellular space within a wound site. The polyfunctional radical scavenger hydrogel formulation is generally formed from a hydrogel, a sterically hindered nitrone and a sterically hindered amine differing from nitrone with respect to at least one property. A portion of the nitrone included with the formulation and/or amine included within the formulation may be dissolved, suspended and/or bonded to a polymer of the hydrogel.

BACKGROUND OF THE INVENTION

Field of the Invention

A polyfunctional radical scavenger hydrogel formulation providingextended protection of the extracellular space capable of assistingwounded tissue in transitioning from the inflammation phase to theproliferation phase of wound healing.

Description of Related Art

Wound healing is an ordered process consisting of four coordinatedphases. In the first phase, hemostasis, fibroblasts and platelets arerecruited to the site of injury to control bleeding through theformation of a clot. The clot releases various cytokines and growthfactors, thereby sending signals to other cells to facilitate healing asthey are programmed to do. For example, cytokines released from the clotrecruit leukocytes, cells of the immune system, to the wound thatinitiate the next phase of wound healing, inflammation.

Inflammation is the second phase of healing. It is considered by many tobe the most unpleasant, as it is associated with many negativeresponses. For instance, inflammation is marked by swelling, redness,irritation, throbbing pain and itchiness. Despite its unpleasantperception, inflammation is very important for proper wound healing asit is during this phase that the wound is cleared of devitalized and/ornecrotic tissue, foreign debris and infectious organisms. In addition toprotecting and cleaning the wound, inflammation also makes the woundready to be repaired.

After being made ready for repair by the inflammation phase, woundhealing enters the third phase, proliferation. It is during this phasethat the collagen extracellular matrix, which acts as a scaffolding tosupport new cells, is laid. New cells produced during the proliferationphase utilize the extracellular matrix scaffolding to migrate into thewound. Eventually a sufficient amount of newly-formed connective tissueand blood vessels, known as granulation tissue, migrates across thecollagen rich extracellular matrix to cover the wound. This migration ofgranulation tissue into the wound allows for the migration of newepithelial cells into the wound to replace lost tissue.

The wound then enters the remodeling phase during which time newlycreated tissue is slowly increased in strength and transformed intomature tissue.

While the phases of wound healing are designed to progress in an orderedcycle, with each phase instigating the next via the release of cytokinesand growth factors, wounds can become stalled in, or regress backwardsto, the inflammation phase. This is quite problematic as theinflammation phase is quite destructive. As mentioned above, theinflammation phase makes the wound ready for repair by cleaning thewound of debris and infectious organisms. A key cleaning agent utilizedby neutrophil and macrophage leukocytes during the inflammation phase isreactive oxygen species.

Reactive oxygen species are very reactive oxygen containing molecules,including superoxide radicals and hydroxyl radicals. Neutrophils, aninitial player in the inflammation phase, release reactive oxygenspecies to kill infectious organisms and breakdown debris. The reactiveoxygen species released by neutrophils, however, are not smart bombs.Rather, reactive oxygen species indiscriminately damage whatever theycome across. Consequently, reactive oxygen species fired off byneutrophils destroy infectious organisms and debris along with healthycells.

Another function of the reactive oxygen species released by neutrophilsis to signal macrophages to enter the wound site. Macrophages, as withneutrophils, utilize reactive oxygen species to kill infectiousorganisms. Generally, they do so by engulfing an infectious organism,and then once inside the macrophage, attacking it with internal storesof reactive oxygen species. If macrophages receive a sufficient signalfrom neutrophils, they release reactive oxygen species in a carpetbombing oxidative burst.

The release of indiscriminately destructive reactive oxygen speciesgenerally is more beneficial than harmful. However, if wound healingbecomes stalled during the inflammation phase, or regresses backwards tothe inflammation phase, the harm caused by the reactive oxygen speciesbombardment of the wound site may quickly outweigh the good. Forexample, the reactive oxygen species may destroy the collagenextracellular matrix scaffolding, thereby inhibiting the migration ofnew tissue into the wound. Additionally, newly grown cells and healthytissue may be damaged and killed by the reactive oxygen species firedinto the wound site.

Accordingly, the excessive release of indiscriminately destructivereactive oxygen species from a prolonged inflammation phase can inhibitwound healing. The destructive power of reactive oxygen species,however, is necessary for neutrophils and macrophages to clear the woundof debris and infectious organisms and otherwise make the wound repairready. Inflammation, therefore, is necessary for proper wound healing.The inflammation phase does, however, need to be limited before itsdetrimental collateral damage begins to outweigh its positive benefits.Accordingly, when a wound has become stalled in, or falls back into, theinflammation phase it is necessary to assist wound healing intransitioning from the inflammation phase to the proliferation phase.

SUMMARY OF THE INVENTION

A polyfunctional radical scavenger hydrogel formulation generallycomprising a hydrogel polymer, a first radical scavenger and a secondradical scavenger, wherein the first radical scavenger and the secondradical scavenger differ with respect to at least one property, mayprovide extended protection of the wound site and thus assist woundedtissue in transitioning from the inflammation phase to proliferationphase of wound healing.

A wound site is divided into two general areas. The first area is theextracellular space. Across lipid cellular membranes within the woundsite is the second area, the intracellular space. Reactive oxygenspecies are generally produced within the intracellular space andreleased into the extracellular space. Once released into theextracellular space, reactive oxygen species may prolong theinflammation phase of wound healing through several mechanisms. Forinstance, reactive oxygen species released into the extracellular spacemay attract macrophages to the wound site that could release additionalreactive oxygen species, thereby increasing the amount of reactiveoxygen species within the wound site. Released reactive oxygen speciesmay also damage and degrade the extracellular matrix thereby deprivingwounds of the necessary scaffolding upon which to rebuild and heal.Lipid membranes may also be damaged by reactive oxygen species, therebycompromising cells and/or triggering apoptosis (programmed cell death).

Radical scavengers are capable of protecting a wound site from thedamaging effects of reactive oxygen species by removing reactive oxygenspecies such as, but not limited to, superoxide radicals and/or hydroxylradicals and other free radicals. Additionally damaging cascadesinitiated by reactive oxygen species may also be lessened and/orarrested by radical scavengers. To elicit such protection, radicalscavengers must have sustained access to the region of the wound siteadversely affected by released reactive oxygen species.

Though comprising two general areas, the extracellular and intracellularspace, a wound site is a complex environment containing a mixture ofregions not equally accessible to all types of molecules. The ability ofa radical scavenger to access the various regions within a wound site isdependent upon its properties, such as solubility and molecular weight(i.e. size) to name a few. The inability of a large radical scavenger toreadily cross lipid membranes separating the intracellular andextracellular space, for example, may limit its access to theintracellular space. Smaller radical scavengers, however, may morereadily cross lipid membranes and thus have the ability to access theintracellular space and the extracellular space. The solubility of aradical scavenger may also influence its ability to access the variousregions of the wound site adversely affected by reactive oxygen species.For instance, a hydrophilic radical scavenger (i.e. soluble in water)may have limited access to lipid membranes and/or the internal regionsof proteins adversely affected by reactive oxygen species. Even if aradical scavenger can access a region adversely affected by reactiveoxygen species, its ability to provide protection may be limited by itsrate of diffusion or movement. A radical scavenger having a high rate ofdiffusion may not remain within an adversely effected region asufficient amount of time to provide adequate protection. Accordingly, apolyfunctional radical scavenger hydrogel formulation comprising a firstradical scavenger and a second radical scavenger, wherein the firstradical scavenger and the second radical scavenger differ with respectto at least one property, may assist a wound in transitioning from theinflammation phase by providing extended protection to various regionswithin the wound site.

The first radical scavenger and the second radical scavenger may differin various properties such as, but not limited to, solubility,reactivity with various reactive oxygen species, affinity for variousreactive oxygen species, stability and molecular weight. The propertiespossessed by the first radical scavenger and the second radicalscavenger control and limit their therapeutic efficacy and duration ofaction within different regions of the wound site. The properties of thefirst radical scavenger may also influence the therapeutic efficacy andduration of action of the second radical scavenger within variousregions of the wound site adversely affected by reactive oxygen species.Accordingly, a preferred polyfunctional radical scavenger hydrogelformulation comprises a first radical scavenger having a certain set ofproperties and a second radical scavenger having a second set propertiesincluding at least one property that differs from that of the firstradical scavenger, as to provide extended protection to various regionsof the wound site adversely affected by reactive oxygen species.

The molecular weight of a radical scavenger may influence the abilityand/or rate at which the radical scavenger exits and enters a regionwithin the wound site adversely affected by reactive oxygen species.Radical scavengers having a small molecular weight, for instance, maymore readily diffuse away from adversely affected regions within thewound site and/or cross cellular membranes. However, if the firstradical scavenger and the second radical scavenger readily exitedadversely affected regions by diffusing away and/or crossing membranes,then the region would be increasingly unprotected from reactive oxygenspecies as more of the radical scavengers exited the region.Accordingly, in some embodiments the polyfunctional radical scavengerhydrogel formulation may provide extended protection by including afirst radical scavenger having a smaller molecular weight than a secondradical scavenger. The molecular weight of at least one of the radicalscavengers in some embodiments may be sufficiently large as to severelylessen diffusion of the radical scavenger.

Though generally aqueous, a wound site contains non-aqueous regions suchas cellular membranes. As such, extended protection of the wound sitemay be provided in some embodiments of the polyfunctional radicalscavenger hydrogel formulation with radical scavengers capable ofaccessing the aqueous and non-aqueous regions of the extracellularspace. Accordingly, in some embodiments, the first radical scavenger maybe more lipid soluble than the second radical scavenger.

Nitrones having a lipid soluble aromatic moiety, for example, may besufficiently lipid soluble to have access to non-aqueous regions of thewound site adversely affected by reactive oxygen species, such as lipidmembranes. Reactive oxygen species can adversely affect cellularmembranes by oxidizing lipids. Cellular membranes are walls utilized bycells to protect themselves from their surroundings and to holdthemselves together. The basic building blocks of the cellular membranesare lipids. Oxidation of the lipid building blocks of the cellularmembrane can create holes within the membrane permitting furthercellular damage. Additionally, oxidized lipids can decompose into lipidacyls, detergents which dissolve the cellular membrane. Accordingly,indiscriminate damage by reactive oxygen species to the lipid buildingblocks of a cellular membrane can destroy the cellular membrane.

The indiscriminate damage of cellular membranes can be amplified by thedamaged lipids themselves. The oxidation of a lipid within a cellularmembrane by a reactive oxygen species produces a lipid radical withinthe membrane. Lipid radicals within the cellular membrane can then befurther oxidized by oxygen and transformed into a lipid peroxyl radical.The resulting lipid peroxyl can then attack another lipid within themembrane, resulting in an additional lipid peroxyl radical which canthen attack another lipid. Each of these peroxyl radicals can decomposeinto a lipid acyl detergent capable of dissolving the cellular membrane.Accordingly, a single reactive oxygen species indiscriminately damaginga lipid can lead to a cascade of damage causing a catastrophic failureof the cellular membrane of a healthy cell.

A cascade of lipid damage may be lessened and/or arrested by nitrones.Accordingly, nitrones having an aromatic moiety making the nitronesufficiently lipid soluble to be able to enter a cellular membranerepresents one potentially preferred class of radical scavengers. As toinhibit transfer of the nitrone across the membrane, and thereby extendprotection of the wound site, it is preferable that the aromatic moietyof the nitrone is not readily susceptible to passive, active and/orfacilitated membrane transport (herein after simply referred to ascellular uptake) across at least one cellular membrane within the woundsite. Accordingly, nitrones having an aromatic moiety not readilysusceptible to cellular uptake across at least one cellular membranewithin the wound site represent one potentially preferred class ofradical scavengers. In some embodiments of the polyfunctional radicalscavenging hydrogel formulation, a first radical scavenger comprising anitrone having a lipid soluble aromatic moiety not readily susceptibleto cellular uptake may be incorporated with a second radical scavengersusceptible to cellular uptake.

Reactive oxygen species present within the wound site may be diverse,including more than one reactive oxygen species. Extended protectionwithin the wound site, therefore, may be provided with a first radicalscavenger which more readily removes at least one of the reactive oxygenspecies present within the wound site than the second radical scavenger.Accordingly, in some embodiments, the first radical scavenger may morereadily react with a given reactive oxygen species than the secondradical scavenger.

Hydroxylamines are particularly reactive towards reactive oxygen speciesand thus represent one potentially preferred class of compounds forradical scavengers to be included within the polyfunctional radicalscavenger hydrogel formulation. Hydroxylamines remove reactive oxygenspecies by first being oxidized by a reactive oxygen species to anitroxide (NO.).

Hydroxylamines may provide extended protection of the extracellularspace of the wound site by intercepting and neutralizing reactive oxygenspecies within the aqueous regions of the wound site that wouldindiscriminately damage healthy tissue and/or attract macrophages to thewound site. Various hydroxylamines, and their corresponding nitroxidederivatives, however, may be to polar (i.e. not sufficiently lipidsoluble) too access and protect some regions within the wound siteadversely affected by reactive oxygen species, such as the non-aqueousinterior of lipid membranes and/or proteins. Accordingly, whilehydroxylamines can intercept and neutralize reactive oxygen species thatwould indiscriminately damage healthy tissue, a given hydroxylamine maynot be able to provide protection to all regions of the wound siteadversely affected by reactive oxygen species. In some embodiments ofthe polyfunctional radical scavenger hydrogel formulation, therefore,the protection provided may be extended by incorporating into thehydrogel formulation a water soluble hydroxylamine and a lipid solubleradical scavenger.

In some embodiments, protection against the adverse effects of reactiveoxygen species within the wound site may be extended, at leastpartially, due to a first radical scavenger which is more reactivetowards at least one reactive oxygen species than a second radicalscavenger kinetically protecting the second radical scavenger fromdegradation by a reactive oxygen species. Various hydroxylamines areparticularly reactive towards reactive oxygen species and thushydroxylamines represent one potentially preferred class of compoundsfor a first radical scavenger that may kinetically protect a secondradical scavenger, such as nitrones.

In the presence of reactive oxygen species a nitrone may be transformedinto a nitroxide (NO.) capable of scavenging radicals. According to onepotential mechanism, the nitroxide formed from the nitrone can bedegraded by a hydrolysis reaction to a hydroxylamine. Accordingly,nitrones may be transformed into nitroxides and then degraded intohydroxylamines by reactive oxygen species. It is also possible thatnitrones within the wound site may be transformed into a hydroxylamineby a hydrolysis reaction when not in the nitroxide state. Ashydroxylamines readily react with reactive oxygen species, when ahydroxylamine and a nitrone are incorporated into a polyfunctionalradical scavenger hydrogel formulation the probability of reactiveoxygen species reacting with the nitrone may be decreased such that thehydroxylamine kinetically protects the nitrone from degradation.Nitrones thus represent one class of radical scavengers that may bekinetically protected by hydroxylamines.

It is possible that the kinetic protection provided by a hydroxylamine,acting as a first radical scavenger, to a nitrone, acting as a secondradical scavenger, may delay degradation following transformation of thenitrone into a nitroxide, thereby potentially extending the protectionof the wound site in some embodiments of the polyfunctional radicalscavenger hydrogel formulation.

Degradation into a hydroxylamine of a nitrone acting as a second radicalscavenger does not completely eliminate the ability of the nitrone toscavenge radicals. As previously discussed, hydroxylamines scavengereactive oxygen species. Accordingly, the degradation of nitrones actingas a second radical scavenger, into hydroxylamines may extend protectionof the wound site by forming a third radical scavenger capable ofremoving reactive oxygen species that would indiscriminately damageand/or attract macrophages to the wound site.

The radical scavengers may be incorporated into the polyfunctionalradical scavenger hydrogel formulation in a variety of manners.Accordingly, in some embodiments all or a portion of the radicalscavengers incorporated into the hydrogel formulation may be dissolvedand/or suspended in the hydrogel. Having at least a portion of theradical scavengers dissolved and/or suspended in the hydrogel may allowfor an initial rapid removal of reactive oxygen species and/or otherradicals from the treated wound site.

In some embodiments, all or a portion of the radical scavengersincorporated into the hydrogel formulation may be bonded to a polymer ofthe hydrogel. Additionally, in some embodiments all or a portion of theradical scavengers incorporated into the hydrogel formulation may bebonded to a molecule that is free of and/or bonded to a polymer of thehydrogel. In some embodiments, bonding the radical scavengers to apolymer of the hydrogel and/or another molecule may provide extendedprotection within the extracellular space of the wound site by limitingthe diffusion of the radical scavengers away from the extracellularspace of the wound site and/or cellular uptake into the intracellularspace within the wound site. In combination or the alternative, theextended protection may result from radical scavengers bonded to apolymer of the hydrogel and/or another molecule being released over timefrom the polymer of the hydrogel and/or the other molecule as to replacepreviously spent radical scavengers.

In some embodiments a first radical scavenger and a second radicalscavenger bonded to a polymer of the hydrogel and/or another moleculemay remove radicals when so bound. In such embodiments, the spacingshould be such that the first radical scavenger does not inhibit and/orinterfere with the ability of the second radical scavenger to removereactive oxygen species. Accordingly, in some embodiments of thepolyfunctional radical scavenger hydrogel formulation at least tworadical scavengers are bonded to a polymer of the hydrogel and/oranother molecule such that the polymer of the hydrogel and/or the othermolecule acts as a spacer molecule. When, in such embodiments, thesecond radical scavenger is intended to provide protection to a regionof the wound site different than the first radical scavenger, thespacing should preferably be such that the first radical scavenger doesnot inhibit or interfere with the ability of the second radicalscavenger to access the intended region of the wound site. For instance,when the first radical scavenger and the second radical scavenger differwith respect to lipid and/or water solubility and are intended toscavenge radicals from different areas of the wound site, the spacingshould be such that the water soluble nature of the first radicalscavenger does not prevent the second radical scavenger from accessingnon-aqueous regions of the wound site and the lipid soluble nature ofthe second radical scavenger does not prevent the ability of the firstradical scavenger to access aqueous regions of the wound site.

As previously mentioned, extended protection of the wound site may beprovided by pairing a more reactive first radical scavenger with asecond radical scavenger such that the first radical scavengerkinetically protects the second radical scavenger from degradation. Suchkinetic protection may be achieved when all or a portion of the firstradical scavenger incorporated into the hydrogel formulation and/orsecond radical scavenger incorporated into the hydrogel formulation arebonded to a polymer of the hydrogel and/or another molecule. When such apairing of a first radical scavenger and a second radical scavenger arebonded to a polymer of the hydrogel and/or another molecule, it ispreferred that the first radical scavenger and the second radicalscavenger are sufficiently close such that the first radical scavengercan protect the second radical scavenger from being degraded by at leastone reactive oxygen species within the wound site.

In some embodiments the protection of the extracellular space of thewound site may be extended by bonding a first radical scavenger and asecond radical scavenger to a polymer of the hydrogel and/or anothermolecule such that the first radical scavenger and the second radicalscavenger have zones of influence that are partially overlapping so asto be partially distinct. In such embodiments, the partially distinctnature of the zone of influence of the second radical scavenger mayprovide the second radical scavenger with an area within its zone ofinfluence relatively unaffected by the presence of the first radicalscavenger. This may facilitate the second radical scavenger accessingregions within the wound site not readily accessible to the firstradical scavenger. At the same time, the portion of the second radicalscavenger's zone of influence affected by the first radical scavengermay permit the first radical scavenger to kinetically protect the secondradical scavenger from being degraded by at least one reactive oxygenspecies within the wound site.

Scavenging nitroxides derived from nitrones may, be on potentialmechanism, be degraded by hydrolysis into hydroxylamines. In combinationor the alternative, nitrones may be directly degraded via hydrolysisinto hydroxylamines. As hydroxylamines are relatively incapable ofscavenging lipid radicals within cellular membranes, the degradation ofthe nitroxide and/or nitrone to a hydroxylamine prevents the nitroneincorporated into the formulation as radical scavenger from providingprotection to cellular membranes indiscriminately damaged by reactiveoxygen species. However, placing a hydroxylamine near a nitrone maykinetically protect the nitroxide derived from the nitrone fromdegradation, thereby potentially extending the protection by the nitroneof the non-aqueous regions of the wound site. Accordingly, theprotection of non-aqueous regions of the wound site may be extended insome embodiments by bonding a nitrone radical scavenger and ahydroxylamine radical scavenger to a molecule so as to protect thederived nitroxide and/or native nitrone from degradation and thusprolong the protection provided by incorporation of the nitrone into theformulation. In some embodiments this protection may be achieved byplacing a hydroxylamine incorporated as a first radical scavengersufficiently close to a nitrone incorporated as a second radicalscavenger to extend the half life of the nitrone.

It is also possible that the partially overlapping zones of influence ofa hydroxylamine, as first radical scavenger, and a nitrone, as a secondradical scavenger, may extend the protection of the wound site by makingthe nitrone more efficient at protecting non-aqueous regions of theextracellular matrix. In this scenario, the faster reaction kinetics ofthe hydroxylamine with various reactive oxygen species may cause theslower reacting nitrone to kinetically favor scavenging lipid radicals.

It is also possible that the fast reaction kinetics of a hydroxylamineacting as a first radical scavenger may extend protection of the woundsite by shielding cellular membranes form further damage while a nitroneacting as a second radical scavenger lessens and/or arrests damagingcascades within the lipid membrane.

Spacing the first radical scavenger and the second radical scavenger asufficient distance as to provide partially overlapping zones ofinfluence can be accomplished by bonding the first radical scavenger toa first end of a spacer molecule and the second radical scavenger to thesecond end of the spacer molecule. In combination or the alternative,spacing the first radical scavenger and the second radical scavenger asto provide partially overlapping zones of influence can be accomplishedby bonding the first radical scavenger and the second radical scavengerto a polymer of the hydrogel. In some embodiments, the derivativescavenging site of the first radical scavenger and the derivativescavenging site of the second radical scavenger may be separated by atleast four bonds. The bonds separating the derivative scavenging site ofthe first radical scavenger and the derivative scavenging site of thesecond radical scavenger may be any combination of single, resonant,double or triple bonds. The derivative scavenging site of a radicalscavenger is the portion of the radical scavenger forming a derivativethat scavenges radicals. Accordingly, in the case of a hydroxylamine, aderivative scavenging site is the NOH functional group, which formsnitroxide (NO.). In the case of a nitrone, a derivative scavenging siteis the NO⁻ portion of the C═NO⁻ functional group, which forms nitroxide(NO.).

In some embodiments, a molecule comprising a first radical scavenger anda second radical scavenger bonded to different ends of a spacer moleculemay be carried upon a polymer of the hydrogel. Such a molecule may bependant to the polymer of the hydrogel. In combination or thealternative, the molecule may be incorporated into a polymer of thehydrogel as a monomer.

The above potential beneficial effects may be wholly or partiallyinduced by spacing a first radical scavenger a sufficient distance froma second radical scavenger such that the first scavenger influences thereaction kinetics of the second scavenger and provides the first radicalscavenger and the second radical scavenger with partially overlappingzones of influence.

While the above potential beneficial effects have been explained withreference to hydroxylamines and nitrones, this has been done merely forpurposes of illustration. Other radical scavengers may be substitutedfor hydroxylamines and/or nitrones to provide all or a portion of theabove described benefits, provided the radical scavengers chosen providea similar pairing of comparative properties necessary to induce thepotential beneficial effect desired.

Radical scavengers may be incorporated into the polyfunctional radicalscavenger hydrogel formulation in virtually any amount. In someembodiments, the polyfunctional radical scavenger hydrogel may comprise10% by mass of radical scavengers. The percent by mass of the firstradical scavenger may be equal to, less than or greater than that of thesecond radical scavenger.

The hydrogel of the polyfunctional radical scavenger hydrogelformulation provides the wound to be treated with the positive benefitsof a hydrogel wound dressing. For instance, the presence of the hydrogelmay absorb exudates, inhibit the formation of the biofilm, maintain thewound in a moistened state and/or provide a matrix for other potentiallybeneficial therapeutic agents. Therapeutic agents that may be suspendedin the hydrogel matrix include atomic silver and silver salts. In someembodiments a quaternary ammonium salt such as benzalkonium chloride orcetylpyridinum chloride may be incorporated as an antibiotic.

The hydrogel may also be utilized to provide an extracellular matrix forthe migration and support of new cells. When the hydrogel is to be soused, the hydrogel polymers may wholly or partially comprise collagen.Hydrogel polymers partially comprising collagen may include collagen asa block polymer or copolymer. In combination or the alternative,hydrogel polymers partially comprising collagen may include collagenpendant groups.

In addition to facilitating the hydrogel providing an extracellularmatrix, the inclusion of collagen within the hydrogel may facilitateassociation with the existing extracellular matrix and/or tissues withinthe wound site. In embodiments in which at least one radical scavengeris carried on a polymer of the hydrogel, this may result in the radicalscavenger carried on the hydrogel being in closer proximity to thetissues of the wound, as to extend protection of the wound site byproviding better protection against indiscriminate damage by reactiveoxygen species. The proximity of a radical scavenger may also beimproved by carrying a radical scavenger on a collagen molecule.

Embodiments of the polyfunctional radical scavenger hydrogel formulationmay be formulated for use as wound ointments. In combination or thealternative, embodiments of the polyfunctional radical scavengerhydrogel formulation may be formulated for use in wound dressings.

As wounds may also occur during catheterization and the introduction ofother medical devices into the body, embodiments of the polyfunctionalradical scavenging hydrogel formulation may be formulated to be used ascoatings on catheters or other medical or dental devices, includingstents, artificial valves, organs and organ parts, pulmonary filters andatrial appendage occlusions devices. The polyfunctional radicalscavenging hydrogel formulations may also be used for administrationdirectly to tissues during surgical procedures where wounds are incurredand/or repaired such as bone or tooth fractures, dental root canalprocedures and visceral soft tissue repairs where indwelling reservoirsof polyfunctional radical scavenging hydrogel formulations may be usedto inhibit the development of excessive inflammation and oxidativestress, such as may occur during tissue repairs, transplantations andimplantations.

It should be appreciated that the foregoing and subsequent references tofirst and second radical scavenger is not to imply or suggest that apolyfunctional radical scavenger hydrogel formulation is limited toincluding only two radical scavengers. A polyfunctional hydrogelformulation may include any number of radical scavengers. It ispreferred, however, that at least two of the radical scavengers differwith respect to at least one property.

DETAILED DESCRIPTION OF THE INVENTION

The polyfunctional radical scavenger hydrogel formulation generallycomprises a hydrogel polymer, a first radical scavenger and a secondradical scavenger. At least a portion of the first radical scavengerand/or the second radical scavenger may be dissolved, suspended and/orotherwise incorporated into the hydrogel formulation without beingbonded to and/or carried on a polymer of the hydrogel. In someembodiments, at least a portion of the first radical scavengerincorporated into the hydrogel formulation and/or the second radicalscavenger incorporated into the hydrogel formulation may be bonded tothe hydrogel polymer and/or to a molecule other than a polymer of thehydrogel. In some embodiments the hydrogel polymer and/or anothermolecule to which a portion of the first radical scavenger incorporatedinto the hydrogel formulation and a portion of the second radicalscavenger incorporated into the hydrogel formulation are bonded may actas a spacer molecule.

In embodiments in which a first radical scavenger and a second radicalscavenger are bonded to a spacer molecule, the first radical scavengerand the second radical scavenger may be sufficiently spaced such thatthe hydrophilic nature of the first radical scavenger does not preventthe second radical scavenger from accessing non-aqueous regions of thewound site and the lipid soluble nature of the second radical scavengerdoes not prevent the first radical scavenger from accessing aqueousregions of the wound site. In combination or the alternative, the firstradical scavenger and the second radical scavenger may be sufficientlyspaced such that the first radical scavenger kinetically protects thesecond radical scavenger. In some embodiments in which a first radicalscavenger and a second radical scavenger are bonded to a spacermolecule, the first radical scavenger and the second radical scavengermay be separated a sufficient distance as to provide the first radicalscavenger with a zone of influence partially overlapping the zone ofinfluence of the second radical scavenger.

The first radical scavenger may be any radical scavenger capable ofscavenging reactive oxygen species. Preferably the first radicalscavenger comprises a sterically hindered amine. An amine can besterically hindered by incorporation of a tertiary carbon adjacent tothe nitrogen of the amine. The sterically hindered amine may be aprimary amine, a secondary amine, a tertiary amine and/or a cyclicamine.

Primary and secondary sterically hindered amines that may be utilized inthe polyfunctional radical scavenger hydrogel formulation can beN-tert-butylamine derivatives in accordance with general formula 1.

where R¹⁻¹, R²⁻¹ and R³⁻¹ are each a carbon chain having a length of C1to C12,

where X¹⁻¹, X²⁻¹ and X³⁻¹ are each selected from H, OH, acrylate,methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide,

where X⁴⁻¹ is selected from H, OH, O and O., and

where R⁴⁻¹ is selected from H and a carbon chain having a length of C1to C12.

Carbon chains at R¹⁻¹, R²⁻¹, R³⁻¹ and R⁴⁻¹ may comprise linear, branchedand/or cyclic portions. Cyclic portions of the carbon chains at R¹⁻¹,R²⁻¹, R³⁻¹ and R⁴⁻¹ may be aromatic. In some embodiments any of thecarbon chains at R¹⁻¹, R²⁻¹, R³⁻¹ and R⁴⁻¹ may be substituted.Embodiments are also possible in which a carbon of any of the carbonchains R¹⁻¹, R²⁻¹, R³⁻¹ and R⁴⁻¹ is replaced with another atom as tocreate a heterogeneous chain.

For example, the carbon chain at R³⁻¹ could be a methyl phenyl and X³⁻¹could be H as to provide a first radical scavenger comprising asterically hindered amine in accordance with general formula 1.1

where R¹⁻¹ and R²⁻¹ are each a carbon chain having a length of C1 toC12,

where X¹⁻¹ and X²⁻¹ are each selected from H, OH, acrylate,methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide,

where X⁴⁻¹ is selected from H, OH, O and O., and

where R⁴⁻¹ is selected from H and a carbon chain having a length of C1to C12.

In some embodiments the phenyl ring of general formula 1.1 may besubstituted as to increase lipid solubility or water solubility.Embodiments are also possible in which the phenyl ring of generalformulation 1.1 is substituted to inhibit or potentiate cellular uptakeacross at least one membrane within the wound site. In some embodiments,a carbon of the phenyl ring of general formula 1.1 may be replaced withanother atom as to create a heterogeneous aromatic ring. The phenyl ringof general formula 1.1 maybe fused and/or conjugated with anotheraromatic ring in some embodiments.

Cyclic sterically hindered hydroxylamines that may be utilized in thepolyfunctional radical scavenger hydrogel formulation can be derivedfrom pyrrole derivatives in accordance with general formula 2.

where R¹⁻², R²⁻², R³⁻² and R⁴⁻² are each a carbon chain having a lengthof C1 to C12,

where X¹⁻², X²⁻², X³⁻², X⁴⁻² and X⁵⁻² are each selected from H, OH,acrylate, methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide, and

where X⁶⁻² is selected from H, OH, O and O..

Carbon chains at R¹⁻², R²⁻², R³⁻² and R⁴⁻² may comprise linear, branchedand/or cyclic portions. Cyclic portions of the carbon chains at R¹⁻²,R²⁻², R³⁻² and R⁴⁻² may be aromatic. In some embodiments any of thecarbon chains at R¹⁻², R²⁻², R³⁻² and R⁴⁻² may be substituted.Embodiments are also possible in which a carbon of any of the carbonchains R¹⁻², R²⁻², R³⁻² and R⁴⁻² is replaced with another atom as tocreate a heterogeneous chain.

Cyclic sterically hindered hydroxylamines that may be utilized in thepolyfunctional radical scavenger hydrogel formulation can also bederived from piperidine derivatives in accordance with general formula3.

where R¹⁻³, R²⁻³, R³⁻³ and R⁴⁻³ are each a carbon chain having a lengthof C1 to C12,

where X¹⁻³, X²⁻³, X³⁻³, X⁴⁻³ and X⁵⁻³ are each selected from H, OH,acrylate, methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide, and

where X⁶⁻³ is selected from H, OH, O and O..

Carbon chains at R¹⁻³, R²⁻³, R³⁻³ and R⁴⁻³ may comprise linear, branchedand/or cyclic portions. Cyclic portions of the carbon chains at R¹⁻³,R²⁻³, R³⁻³ and R⁴⁻³ may be aromatic. In some embodiments any of thecarbon chains at R¹⁻³, R²⁻³, R³⁻³ and R⁴⁻³ may be substituted.Embodiments are also possible in which a carbon of any of the carbonchains R¹⁻³, R²⁻³, R³⁻³ and R⁴⁻³ is replaced with another atom as tocreate a heterogeneous chain.

When any of X¹⁻¹, X²⁻¹ and X³⁻¹ in general formula 1, or any of X¹⁻²,X²⁻², X³⁻², X⁴⁻² and X⁵⁻² in general formula 2, or any X¹⁻³, X²⁻³, X³⁻³,X⁴⁻³ and X⁵⁻³ in general formula 3, are any of the functional groups OH,acrylate, methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide, the radical scavenger may be joined to thehydrogel polymer and/or another molecule by X¹⁻¹, X²⁻¹ or X³⁻¹, in thecase of general formula 1, or X¹⁻², X²⁻², X³⁻², X⁴⁻² or X⁵⁻² in the caseof general formula 2, or X¹⁻³, X²⁻³, X³⁻³, X⁴⁻³ or X⁵⁻³ in the case ofgeneral formula 3. When so joined to the hydrogel polymer, the radicalscavenger may be pendant to the hydrogel polymer and/or a monomer of thehydrogel polymer.

The second radical scavenger may be any radical scavenger capable ofscavenging reactive oxygen species. The second radical scavenger may bechosen so as to provide a radical scavenger with properties differentthan that of the first radical scavenger. For example, the secondradical scavenger may have different reaction kinetics with regards toat least one reactive oxygen species than the first radical scavenger.In combination or the alternative, the second radical scavenger may bemore lipid soluble than the first radical scavenger.

The second radical scavenger may also be chosen so as to have radicalscavenging capabilities not possessed by the first radical scavenger.For instance, the second radical scavenger may be more capable ofscavenging lipid radicals than the chosen first radical scavenger.

The second radical scavenger may be capable of being degraded into athird radical scavenger.

Though not necessary, it is preferred that the second radical scavengermeet at least a portion of the above identified criteria. Accordingly apreferred second radical scavenger is a sterically hindered nitrone.Sterically hindered nitrones that may be utilized in the polyfunctionalradical scavenger hydrogel formulation can be derived from arylN-tert-butylnitrone derivatives in accordance with the general formula4.

where R¹⁻⁴, R²⁻⁴, and R³⁻⁴ are each a carbon chain having a length of C1to C12,

where X¹⁻⁴, X²⁻⁴, and X³⁻⁴ are each selected from H, OH, acrylate,methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide, and

where Y is an aromatic moiety comprising an aromatic ring having asubstituent containing a carbon that is bonded to the nitrogen by thedouble bond.

In some embodiments, the aromatic moiety Y in general formula 4 may berepresented by the general formula:

where R^(Y) is a substituent having a carbon that is bonded to thenitrogen of general Formula 4 by the double bond

Carbon chains at R¹⁻⁴, R²⁻⁴ and R³⁻⁴ may comprise linear, branchedand/or cyclic portions. Cyclic portions of the carbon chains at R¹⁻⁴,R²⁻⁴ and R³⁻⁴ may be aromatic. In some embodiments any of the carbonchains at R¹⁻⁴, R²⁻⁴ and R³⁻⁴ may be substituted. Embodiments are alsopossible in which a carbon of any of the carbon chains R¹⁻⁴, R²⁻⁴ andR³⁻⁴ is replaced with another atom as to create a heterogeneous chain.

In some embodiments the aromatic moiety Y of general formula 4 maycomprise one or more substituents increasing lipid solubility and/orlimiting cellular uptake across at least one cellular member within thewound site. For example the aromatic moiety Y of general formula 4 maycomprise a halide containing substituent. In combination of thealternative, a carbon of the aromatic moiety Y may be replaced withanother atom as to create a heterocyclic aromatic moiety. In someembodiments, the aromatic ring of the aromatic moiety Y may be fusedand/or conjugated with another aromatic ring. Preferably, thepolyfunctional radical scavenger hydrogel formulation may comprisederivatives of formula 4 in which the aromatic moiety Y is lipid solubleand not subject to cellular uptake across at least one cellular membranewithin the wound site via active and/or facilitated transport.Accordingly, the aromatic moiety Y may be a methyl phenyl as to producea molecule in accordance with general formula 4.1.

where R¹⁻⁴, R²⁻⁴, and R³⁻⁴ are each a carbon chain having a length of C1to C12, and

where X¹⁻⁴, X²⁻⁴, and X³⁻⁴ are each selected from H, OH, acrylate,methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide.

The lipid solubility of a molecule in accordance with general formula4.1 may also be increased by utilizing a phenyl or another aromaticmoiety for any of R¹⁻⁴, R²⁻⁴ and R³⁻⁴. For instance, selecting as R³⁻⁴ amethyl phenyl and selecting as X³⁻⁴ H, as to provide a molecule inaccordance with general formula 4.2, may provide a radical scavengerhaving improved access to a lipid membrane and/or duration action withina lipid membrane.

where R¹⁻⁴ and R²⁻⁴ are each a carbon chain having a length of C1 toC12, and

where X¹⁻⁴, X²⁻⁴, and X³⁻⁴ are each selected from H, OH, acrylate,methacrylate, N-(alkyl carboxyl)-acrylamide, and N-(alkylcarboxyl)-methacrylamide.

When any of X¹⁻⁴, X²⁻⁴ and X³⁻⁴ in general formula 4 are any of thefunctional groups OH, acrylate, methacrylate, N-(alkylcarboxyl)-acrylamide, and N-(alkyl carboxyl)-methacrylamide, the radicalscavenger may be joined to the hydrogel polymer and/or another moleculeby X¹⁻⁴, X²⁻⁴ or X³⁻⁴. When so joined to the hydrogel polymer, theradical scavenger may be pendant to the hydrogel polymer and/or amonomer of the hydrogel polymer.

In some embodiments, at least a portion of the first radical scavengerincorporated into the hydrogel formulation and a portion of the secondradical scavenger incorporated into the hydrogel formulation are bondedto a spacer molecule. Preferably the spacer molecule separates the firstradical scavenger from the second radical scavenger a sufficientdistance so as to provide the first radical scavenger with a zone ofinfluence partially overlapping the zone of influence of the secondradical scavenger. Spacing the first radical scavenger and the secondradical scavenger a sufficient distance as to provide partiallyoverlapping zones of influence can be accomplished by bonding the firstradical scavenger to a first end of a molecule and the second radicalscavenger to the second end of the molecule. Preferably, the derivativescavenging site of the first radical scavenger and the derivativescavenging site of the second radical scavenger are separated by atleast four bonds. The bonds separating the derivative scavenging site ofthe first radical scavenger and the derivative scavenging site of thesecond radical scavenger may be any combination of single, resonant,double or triple bonds.

In some embodiments the spacer molecule may be bonded to the hydrogelpolymer of the polyfunctional radical scavenger hydrogel formulation. Insuch embodiments, the first radical scavenger and/or second radicalscavenger may be a monomer of the hydrogel polymer, a monomer of a blockpolymer of the hydrogel polymer, and/or a monomer of a copolymer of thehydrogel polymer. In such a situation the hydrogel polymer may besynthesized such that a monomer comprising the first radical scavengeris spaced a sufficient distance along the hydrogel polymer from amonomer comprising the second radical scavenger. In combination or thealternative, the first radical scavenger and/or second radical scavengermay be pendent to the hydrogel polymer.

In addition to the sterically hindered nitrones and sterically hinderedamines corresponding to general formulae 1-4, other radical scavengersmay be utilized in synthesizing embodiments of the polyfunctionalradical scavenger formulation in which at least a portion of the firstradical scavenger and/or second radical scavenger incorporated into thehydrogel formulation are bonded to the polymer of the hydrogel and/oranother molecule.

The polyfunctional radical scavenger may be synthesized frompolyethylene glycols and/or polypropylene glycols monomers carryingpendant nitrones and/or sterically hindered amines. In combination orthe alternative, hydroxyethyl methacrylate and/or hydroxymethyl acryloylchloride carrying nitrones and/or sterically hinder amines may beutilized to incorporate the first and second radical scavengers into thehydrogel polymer. When carried upon hydroxyethyl methacrylate, thearomatic nitrones, sterically hindered amines and/or other radicalscavengers may be incorporated into the polymer by converting thehydroxyethyl methacrylate to glycidic acid.

If a sterically hindered nitrone and/or sterically hinderedhydroxylamine are to be utilized as a scavenger in the polyfunctionalradical scavenger hydrogel formulation, they can be produced from asynthon in accordance with compound A.

where R¹, R² and R³ are carbon chains,

where X¹, X² and X³ are selected from H and OH, and

at least one of X¹, X² and X³ is OH.

Utilization of compound A, or a derivative thereof, as a synthon in theproduction nitrone may be accomplished via a condensation reaction.During such a condensation reaction the carbon chain R¹, R² and/or R³having a hydroxyl may be activated by deprotonation of the hydroxyl orby dehydration of the hydroxyl. The activated carbon chain can then becondensed with a suitable aromatic or heterocyclic aldehyde or ketone togenerate the desired nitrone. The aldehyde or ketone condensed withcompound A may or may not include phenolic, carboxylic or aminosubstituents.

Activation of the carbon chain creates an electrophile at the carbonadjacent the hydroxyl. During condensation, this electrophile can benucleophilically attacked by the carbonyl oxygen of the aldehyde orketone. At the same time, the electrophile carbonyl carbon of thealdehyde or ketone can be nucleophilically attacked by the nitrogen ofthe same or different molecule of compound A. If the aldehyde or ketoneis attacked by the same molecule of compound A, the combinednucleophilic attacks forms a ring intermediate. Deportation of theattacking nitrogen creates a double bond between the nitrogen andcarbonyl carbon of the aldehyde or ketone. At the same time, protonationof the carbonyl oxygen provides the activated carbon chain with ahydroxyl formed from the carbonyl oxygen, thereby forming an alcohol ora carboxylic acid. The amino, carboxyl, phenolic and/or alcoholichydroxyl groups that can be provided by the condensation of compound Awith an aldehyde or ketone provide facile routes to the desired esters,carbamates and amides.

Condensation reactions utilizing compound A, or a derivative thereof, toproduce a nitrone may be accomplished via base catalyzed condensation ina one pot synthetic procedure.

In the case of a C-nitro compound corresponding to the hydroxylamine tobe used, synthons in accordance with compound A can be generated in situby reduction with zinc dust and acetic acid or ammonium chloride.

At least a portion of the first radical scavenger and/or the secondradical scavenger incorporated into the hydrogel formulation may bebonded to a wide variety of molecules, which in some embodiments mayserve as a spacer molecule.

In some embodiments, at least a portion of the first radical scavengerand/or second radical scavenger incorporated into the hydrogelformulation may be bonded to a portion of the hydrogel polymer.

In some embodiments at least a portion of the first radical scavengerand/or second radical scavenger incorporated into the hydrogelformulation may be bonded to a distinct molecule separating thederivative scavenging site of the first radical scavenger from thederivative scavenging site the second radical scavenger.

In some embodiments at least a portion of the first radical scavengerincorporated into the hydrogel formulation and a portion of the secondradical scavenger incorporated into the hydrogel formulation are bondedto a molecule such that the zone of influence of the first radicalscavenger partially overlaps with the zone of influence of the secondradical scavenger.

A spacer molecule distinct from the hydrogel polymer may be formed byR³⁻¹X³⁻¹ of general formula 1 reacting with R²⁻⁴X²⁻⁴ of general formula4. Preferred molecules among the various molecules that may be producedfrom a such reaction include molecules according to general formula 5.—CH₂X¹⁻⁵(CH₂)_(n)X²⁻⁵CH₂—,  Formula 5:

where X¹⁻⁵ and X²⁻⁵ are selected from amide and carboxyl.

Joined first and second radical scavengers formed by the reaction ofR³⁻¹X³⁻¹ of general formula 1 with R²⁻⁴X²⁻⁴ of general formulae 4 as toincorporate a molecule according to general formula 5 includebifunctional radical scavengers represented by general formula 6.

Joined first and second radical scavengers according to general formula6, and others formed by R³⁻¹X³⁻¹ of general formula 1 reacting withR²⁻⁴X²⁻⁴ of general formula 4, may be joined to the hydrogel polymer byX¹⁻¹ or X²⁻¹ of general formula 1 and/or by X¹⁻⁴ or X³⁻⁴ of generalformula 4. Joined first and second radical scavengers according togeneral formula 6 may also be joined to a monomer of a hydrogel polymerby a carbonyl of the molecule according to general formula 5.

A molecule distinct from the hydrogel polymer may likewise be formed byX⁵⁻² of general formula 2 or of X⁵⁻³ general formula 3 reacting withR²⁻⁴X²⁻⁴ of general formulae 4. Preferred molecules among the variousmolecules that may be produced from such a reaction include moleculesaccording to general formula 7.—X¹⁻⁶(CH₂)_(n)X²⁻⁶CH₂—,  Formula 7:

where X¹⁻⁶ and X²⁻⁶ are selected from amide and carboxyl.

Joined first and second radical scavengers formed by X⁵⁻³ of generalformula 3 reacting with R²⁻⁴X²⁻⁴ of general formulae 4 as to incorporatea molecule according to general formula 7 include bifunctional radicalscavengers represented by general formula 8.

Joined first and second radical scavengers according to general formula8, and others formed by X⁵⁻³ of general formula 3 reacting with R²⁻⁴X²⁻⁴of general formulae 4, may be joined to the hydrogel polymer by X¹⁻³,X²⁻³, X³⁻³ or X⁴⁻³ of general formula 3 and/or by X¹⁻⁴ or X³⁻⁴ ofgeneral formula 4. Joined first and second radical scavengers accordingto general formula 8 may also be joined to a monomer of the hydrogelpolymer by a carbonyl of the molecule according to general formula 7.

Joined first and second radical scavengers formed by X⁵⁻³ of generalformula 3 reacting with R²⁻⁴ X²⁻⁴ of general formula 4 as to incorporatea molecule according to general formula 7 also include bifunctionalradical scavengers represented by general formula 9.

Joined first and second radical scavengers according to general formula9, and others formed by X⁵⁻³ of general formula 3 reacting with R²⁻⁴X²⁻⁴of general formula 4, may be joined to the hydrogel polymer by X¹⁻³,X²⁻³, X³⁻³ or X⁴⁻³ of general formula 3 and/or by X¹⁻⁴ or X³⁻⁴ ofgeneral formula 4. Joined first and second radical scavengers accordingto general formula 9 may also be joined to a monomer of the hydrogelpolymer by a carbonyl of the molecule according to general formula 7.

The hydrogel polymer of the polyfunctional radical scavenger hydrogelformulation is not particular limited. Preferred hydrogel polymersinclude block and/or copolymers of collagen, PLURONIC® PF-127 (triblockethylene oxide/propylene oxide), 2-hydroxyethyl methacrylate, acrylate,keratin, pectins, polyvinylpyrrolidones and singly.

Polyfunctional radical scavenger hydrogel formulations generallycomprising a hydrogel, a first radical scavenger and a second radicalscavenger, wherein at least a portion of the first radical scavengerincorporated into the hydrogel formulation and a portion of the secondradical scavenger incorporated into the hydrogel formulation are bondedto the hydrogel, can provide sustained radical scavenging activitylasting up to ninety-six hours.

The ability of a polyfunctional radical scavenger hydrogel formulationto offer extended protection of a wound site over a period of time canbe assessed in the following manner. Neutrophils may be seeded into afirst chamber having a porous surface. After 2 hours, the neutrophilsmay be rinsed with DPBS to remove neutrophils non-adherent to thechamber. The chamber may then be moved to a second chamber containingthe polyfunctional radical scavenger hydrogel formulation to beassessed.

In addition to the polyfunctional radical scavenger hydrogel formulationto be assessed, a fluorescent probe for extracellular reactive oxygenspecies and an fMLP solution, which stimulates neutrophils to producereactive oxygen species, should be added to the second chamber.

After incubation with the probe and stimulate, the activity of thepolyfunctional radical scavenger hydrogel formulation can be observedfor a period of time. After which, supernatants from the first chambermay be removed to a separate well plate for fluorescence detection.

As controls, neutrophils should be identically seeded in controlchambers. The control chambers may be incubated in a second chambercontaining only the probe and stimulant. After incubation with the probeand stimulant for the same period of time as neutrophils exposed to thepolyfunctional radical scavenger hydrogel formulation being assessed,supernatants from the control chamber may be removed to a separate wellplate for fluorescence detection.

An increase in the fluorescence detected over time will indicatesustained levels of reactive oxygen species. Conversely, a decrease inthe fluorescence detected over time indicates sustained scavenging ofreactive oxygen species by the polyfunctional radical scavenger hydrogelformulation being assessed.

A polyfunctional radical scavenger hydrogel formulation generallycomprising a hydrogel, first radical scavenger and a second radicalscavenger, wherein at least a portion of the first radical scavengerincorporated into the hydrogel formulation and a portion of the secondradical scavenger incorporated into the hydrogel formulation are bondedto the hydrogel, can provide immediate and sustained radical scavengingactivity lasting up to ninety-six hours. The immediate and sustainedradical scavenging activity of polyfunctional radical scavenger hydrogelformulation allows such a formulation to be used as coatings on medicaldevices introduced into the body to suppress inflammation and promotetransition into the proliferation phase in wounds generated by theinsertion of the device for a prolonged period of time. Accordingly,polyfunctional radical scavenger hydrogel formulation in accordance withthe present disclosure are beneficial coatings for catheters, stents,artificial valves, organs or organ parts, pulmonary filters, atrialappendage occlusions devices and other medical or dental devices. Thepolyfunctional radical scavenging hydrogel formulations may also be usedfor administration directly to tissues during surgical procedures wherewounds are incurred and/or repaired such as bone or tooth fractures,dental root canal procedures and visceral soft tissue repairs whereindwelling reservoirs of polyfunctional radical scavenging hydrogelformulations may be used to inhibit the development of excessiveinflammation and oxidative stress such as occurs during tissue repairs,transplantations and implantations.

The immediate and sustained radical scavenging activity of bifunctionalradical scavengers carried on hydrogel polymers also makes hydrogelcompositions in accordance with the present disclosure beneficial foruse in wound ointments and wound dressings.

These and other advantages may be realized by polymer compositions inaccordance with the present disclosure and detailed in the followingclaims.

Although the foregoing has been described with reference to potentialmodes of action and/or mechanisms of action for purposes of illustrationand clarity of understanding, it should be appreciated thatpolyfunctional radical scavenger hydrogel formulations in accordancewith the present invention are not limited to the potential modes ofaction and mechanisms of action discussed herein. Polyfunctional radicalscavenger hydrogel formulations within the scope of the invention mayutilize modes of action and/or mechanisms of action in combination withor in the alternative to those discussed herein.

We claim:
 1. A polyfunctional radical scavenger hydrogel comprising: a.a hydrogel; b. a first radical scavenger, the first radical scavengerpresent in an amount providing protection of a surgical wound againstreactive oxygen species that result from hydrogen peroxide within thesurgical wound, the first radical scavenger comprising a stericallyhindered amine of the general formula:

wherein R¹⁻¹ is a carbon chain having a length of C1 to C12, wherein thecarbon chain is linear, branched, cyclic, aromatic portion, or acombination thereof, wherein R²⁻¹ is a carbon chain having a length ofC1 to C12, wherein the carbon chain is linear, branched, cyclic,aromatic portion, or a combination thereof, wherein R³⁻¹ is selectedfrom the group consisting of H and a carbon chain having a length of C1to C12, wherein the carbon chain is linear, branched, cyclic, aromaticportion, or a combination thereof, wherein R⁴⁻¹ is selected from thegroup consisting of H and a carbon chain having a length of C1 to C12,wherein the carbon chain is linear, branched, cyclic, aromatic portion,or a combination thereof, wherein X¹⁻¹ is selected from the groupconsisting of H, OH, acrylate, and methacrylate, wherein X²⁻¹ isselected from the group consisting of H, OH, acrylate, and methacrylate,wherein X³⁻¹ is selected from the group consisting of H, OH, acrylate,and wherein X⁴⁻¹ is selected from the group consisting of H, OH, O andO—; and c. a second radical scavenger; wherein the second radicalscavenger is lipid soluble and wherein the second radical scavenger ispresent in an amount to provide against reactive oxygen species withinthe surgical wounds, the second radical scavenger comprising asterically hindered nitrone of the general formula:

wherein R¹⁻⁴ is a carbon chain having a length of C1 to C12, wherein thecarbon chain is linear, branched, cyclic, aromatic portion, or acombination thereof, wherein R²⁻⁴ is a carbon chain having a length ofC1 to C12, wherein the carbon chain is linear, branched, cyclic,aromatic portion, or a combination thereof, wherein R³⁻⁴ is a carbonchain having a length of C1 to C12, wherein the carbon chain is linear,branched, cyclic, aromatic portion, or a combination thereof, whereinX¹⁻⁴ is selected from the group consisting of H, OH, acrylate, andmethacrylate, wherein X²⁻⁴ is selected from the group consisting of H,OH, acrylate, and methacrylate, wherein X³⁻⁴ is selected from the groupconsisting of H, OH, acrylate, and methacrylate, wherein Y is a lipidsoluble aromatic moiety comprising an aromatic ring having a substituentcontaining a carbon that is bonded to the nitrogen by the double bondand the lipid soluble aromatic moiety inhibits transfer of the nitroneacross a cellular membrane; and wherein the first free radical scavengerkinetically protects and slows the degradation of the second freeradical scavenger and extends the protection of the wound provided bythe polyfunctional radical scavenger hydrogel; and the first freeradical scavenger and the second free radical scavenger are suspended ordissolved within the hydrogel.
 2. The polyfunctional radical scavengerhydrogel of claim 1, wherein the lipid soluble aromatic moiety is notsubject to cellular uptake across at least one cellular membrane withinthe wound.
 3. The polyfunctional radical scavenger hydrogel of claim 2,wherein the second radical scavenger has a higher molecular weight thanthe first radical scavenger.
 4. The polyfunctional radical scavengerhydrogel of claim 1, wherein the first radical scavenger and the secondradical scavenger are suspended in the hydrogel.
 5. The polyfunctionalradical scavenger hydrogel of claim 1, wherein the first radicalscavenger and the second radical scavenger are dissolved in thehydrogel.
 6. The polyfunctional radical scavenger hydrogel of claim 1,wherein the polyfunctional radical scavenger hydrogel compositionprovides extended protection of the extracellular space and assistssurgically wounded tissue in transitioning from the inflammation phaseto the proliferation phase of wound healing.
 7. A hydrogel compositioncomprising: a hydrogel; and a plurality of free radical scavengers thatprovide protection against reactive oxygen species within a wound,wherein the plurality of free radical scavengers consist of: (1) a firstradical scavenger, the first radical scavenger present in an amountproviding protection of the wound against reactive oxygen species withinthe wound, the first radical scavenger comprising a sterically hinderedamine of the general formula:

wherein R¹⁻¹ is a carbon chain having a length of C1 to C12, wherein thecarbon chain is linear, branched, cyclic, aromatic portion, or acombination thereof, wherein R²⁻¹ is a carbon chain having a length ofC1 to C12, wherein the carbon chain is linear, branched, cyclic,aromatic portion, or a combination thereof, wherein R³⁻¹ is selectedfrom the group consisting of H and a carbon chain having a length of C1to C12, wherein the carbon chain is linear, branched, cyclic, aromaticportion, or a combination thereof, wherein R⁴⁻¹ is selected from thegroup consisting of H and a carbon chain having a length of C1 to C12,wherein the carbon chain is linear, branched, cyclic, aromatic portion,or a combination thereof, wherein X¹⁻¹ is selected from the groupconsisting of H, OH, acrylate, and methacrylate, wherein X²⁻¹ isselected from the group consisting of H, OH, acrylate, and methacrylate,wherein X³⁻¹ is selected from the group consisting of H, OH, acrylate,and wherein X⁴⁻¹ is selected from the group consisting of H, OH, O andO—; and (2) a second radical scavenger wherein the second radicalscavenger is lipid soluble and wherein the second radical scavenger ispresent in an amount to provide protection against reactive oxygenspecies within the wound, the second radical scavenger comprising asterically hindered nitrone of the general formula:

wherein R¹⁻⁴ is a carbon chain having a length of C1 to C12, wherein thecarbon chain is linear, branched, cyclic, aromatic portion, or acombination thereof, wherein R²⁻⁴ is a carbon chain having a length ofC1 to C12, wherein the carbon chain is linear, branched, cyclic,aromatic portion, or a combination thereof, wherein R³⁻⁴ is a carbonchain having a length of C1 to C12, wherein the carbon chain is linear,branched, cyclic, aromatic portion, or a combination thereof, whereinX¹⁻⁴ is selected from the group consisting of H, OH, acrylate, andmethacrylate, wherein X²⁻⁴ is selected from the group consisting of H,OH, acrylate, and methacrylate, wherein X³⁻⁴ is selected from the groupconsisting of H, OH, acrylate, and methacrylate, wherein Y is a lipidsoluble aromatic moiety comprising an aromatic ring having a substituentcontaining a carbon that is bonded to the nitrogen by the double bondand the lipid soluble aromatic moiety inhibits transfer of the nitroneacross a cellular membrane.
 8. The hydrogel composition of claim 7,wherein the lipid soluble aromatic moiety is not subject to cellularuptake across at least one cellular membrane within the wound and thefirst free radical scavenger and the second free radical scavenger worksynergistically together to extend the protection of the wound site fromhydrogen peroxide beyond the protection provided by the first freeradical scavenger and the second free radical scavenger individually. 9.The hydrogel composition of claim 8, wherein the second radicalscavenger has a higher molecular weight than the first radicalscavenger.
 10. The hydrogel composition of claim 7, wherein the firstradical scavenger and the second radical scavenger are suspended in thehydrogel and the wound is a surgical wound.
 11. The hydrogel compositionof claim 7, wherein the first radical scavenger and the second radicalscavenger are dissolved in the hydrogel and the wound is a surgicalwound.
 12. The hydrogel composition of claim 10, wherein thepolyfunctional radical scavenger hydrogel composition provides extendedprotection of the extracellular space and assists surgically woundedtissue in transitioning from the inflammation phase to the proliferationphase of wound healing.
 13. The hydrogel composition of claim 11,wherein the polyfunctional radical scavenger hydrogel compositionprovides extended protection of the extracellular space and assistssurgically wounded tissue in transitioning from the inflammation phaseto the proliferation phase of wound healing.
 14. The hydrogelcomposition of claim 7, wherein the polyfunctional radical scavengerhydrogel composition provides extended protection of the extracellularspace and assists surgically wounded tissue in transitioning from theinflammation phase to the proliferation phase of wound healing.
 15. Ahydrogel composition comprising: a hydrogel; and a first radicalscavenger, the first radical scavenger present in an amount providingprotection of the wound against reactive oxygen species that result fromwithin the wound, the first radical scavenger comprising a stericallyhindered amine of the general formula:

wherein R¹⁻¹ is a carbon chain having a length of C1 to C12, wherein thecarbon chain is linear, branched, cyclic, aromatic portion, or acombination thereof, wherein R²⁻¹ is a carbon chain having a length ofC1 to C12, wherein the carbon chain is linear, branched, cyclic,aromatic portion, or a combination thereof, wherein R³⁻¹ is selectedfrom the group consisting of H and a carbon chain having a length of C1to C12, wherein the carbon chain is linear, branched, cyclic, aromaticportion, or a combination thereof, wherein R⁴⁻¹ is selected from thegroup consisting of H and a carbon chain having a length of C1 to C12,wherein the carbon chain is linear, branched, cyclic, aromatic portion,or a combination thereof, wherein X¹⁻¹ is selected from the groupconsisting of H, OH, acrylate, and methacrylate, wherein X²⁻¹ isselected from the group consisting of H, OH, acrylate, and methacrylate,wherein X³⁻¹ is selected from the group consisting of H, OH, acrylate,and wherein X⁴⁻¹ is selected from the group consisting of H, OH, O andO—; and a second radical scavenger wherein the second radical scavengeris lipid soluble and wherein the second radical scavenger is present inan amount to provide protection against reactive oxygen species withinthe wound, the second radical scavenger comprising a sterically hinderednitrone of the general formula:

wherein R¹⁻⁴ is a carbon chain having a length of C1 to C12, wherein thecarbon chain is linear, branched, cyclic, aromatic portion, or acombination thereof, wherein R²⁻⁴ is a carbon chain having a length ofC1 to C12, wherein the carbon chain is linear, branched, cyclic,aromatic portion, or a combination thereof, wherein R³⁻⁴ is a carbonchain having a length of C1 to C12, wherein the carbon chain is linear,branched, cyclic, aromatic portion, or a combination thereof, whereinX¹⁻⁴ is selected from the group consisting of H, OH, acrylate, andmethacrylate, wherein X²⁻⁴ is selected from the group consisting of H,OH, acrylate, and methacrylate, wherein X³⁻⁴ is selected from the groupconsisting of H, OH, acrylate, and methacrylate, wherein Y is a lipidsoluble aromatic moiety comprising an aromatic ring having a substituentcontaining a carbon that is bonded to the nitrogen by the double bondand the lipid soluble aromatic moiety inhibits transfer of the nitroneacross a cellular membrane; and wherein the hydrogel compositionprovides extended protection of the extracellular space and assistssurgically wounded tissue in transitioning from the inflammation phaseto the proliferation phase of wound healing.
 16. The hydrogelcomposition of claim 15, wherein the lipid soluble aromatic moiety isnot subject to cellular uptake across at least one cellular membranewithin the wound.
 17. The hydrogel composition of claim 16, wherein thesecond radical scavenger has a higher molecular weight than the firstradical scavenger.
 18. The hydrogel composition of claim 15, wherein thefirst radical scavenger and the second radical scavenger are suspendedin the hydrogel and the wound is a surgical wound.
 19. The hydrogelcomposition of claim 15, wherein the first radical scavenger and thesecond radical scavenger are dissolved in the hydrogel and the wound isa surgical wound.
 20. The hydrogel composition of claim 15, wherein thefirst free radical scavenger slows the degradation of the second freeradical scavenger.